Techniques using nanopores attract tremendous attentions as an approach to the implementation of post-Next-Generation DNA sequencers. It is considered that the nanopore techniques have a noteworthy advantage in that they allow the characteristics of a biopolymer to be analyzed without DNA labeling, namely using no reagents, such as an enzyme and fluorescent dye. The nanopores may be largely classified into two types. One is a so-called bionanopore, of which channel protein forming a nano-sized aperture (hereinafter, simply referred to as a “nanopore”) is laid inside a bilayer membrane, and another is a so-called solid state nanopore, which is made by microfabricating a semiconductor material.
Two types of DNA analytical methods using these nanopores have been proposed. The first one is a current blockade method. Specifically, ionic current flows through the nanopore when voltage is applied between electrodes, each of which is disposed with electrolyte solution in each of reservoirs for solution installed on the both sides of a thin film with the nanopore formed thereon. The magnitude of the ionic current is proportional to the cross sectional area of the nanopore in terms of the first approximation. When DNA passes through the nanopore, it blocks the nanopore and reduces its effective cross-sectional area, resulting in a reduction of ionic current. The amount of the reduction of ionic current is called current blockade. A difference between a single stranded DNA and a double stranded DNA may be discriminated based on the magnitude of the current blockade. Moreover, it has been reported that one form of nanopore enables the types of DNA bases to be discriminated based on the magnitude of the current blockade (Nonpatent Literature 1, hereinafter also simply referred to as a “first example, according to prior art”). However, it is considered that since the bilayer membrane used for the biopore is a fragile thin film made of weakly-associated low molecular organic compounds, it has a problem of mechanical instability. Moreover, since the process of integrating channel protein inside the bilayer membrane relies on a natural phenomenon, the bilayer membrane has a problem with control of the number of channels and reproducibility. In contrast, it is considered that a solid state nanopore, under which thin film is formed of a semiconductor substrate, or the like, is advantageous in structural stability over a bionanopore. Since the nanopore is mechanically formed, it has another advantage. Furthermore, a device and method for analyzing biomolecules passing through a solid state nanopore formed on a thin film made of graphene instead of the semiconductor substrate have been reported (Patent Document 1). However, identification of the types of DNA bases using the nanopore by means of current blockade has not been reported.
The second one is a tunneling current method. Specifically, the method has been proposed, which is characterized in that a pair of electrodes are disposed facing to each other on the nanopore wall; voltage is applied between the electrodes; DNA passing through the nanopore and tunneling current between the electrodes are measured; and the DNA is analyzed based on the magnitude of the tunneling current. Such a related technique has been reported that when a nucleoside with modified sugar is dissolved in an organic solvent and introduced between nanogap electrodes, and then the tunneling current is measured using a scanning probe microscope, the average of tunneling current values depends on the types of the bases (Nonpatent Document 2, hereinafter, also simply referred to as a “second example, according to prior art”). However, the second example has limitations on experimental conditions because the sample is a nucleoside (containing no phosphoric acid) but not a chained nucleic acid; the sample needs to be modified; the sample needs to be dissolved in an organic solvent; and no nanopore is used, as well as has a problem of low ability to identify bases because the tunneling current has a distribution and partially overlapped between different types of bases.
Other methods for determining base sequences using the nanopore or similar structure have been reported including such methods that a nucleotide (monomer) is separated from a chained nucleic acid (polymer) with an enzyme and caused to pass through a nanochannel or microchannel filled with an aggregate (100 nm to 200 nm in size), for example, an aggregate of silver particulates, and then the nucleotide is identified on the surface of the silver-particulate aggregate by Surface Enhanced Raman Scattering (Patent Document 2); and that an enzyme or the like is filled inside the nanopore to be caused to interact with the nucleotide in a DNA sequence, the resulting bond is controlled using the nanopore, and the nucleotide is determined (Patent Document 3). Any of the aforementioned methods needs to use agents, such as an enzyme, and has complicated device construction and processes.
On the other hand, TERS (Tip Enhanced Raman Scattering) has been reported as another approach to the measurement of a single molecular nucleic acid without labeling (Nonpatent Document 3, hereinafter, also simply referred to as a “third example, according to prior art”). According to this method, a silver tip is formed on the tip of an AFM (Atomic Force Microscope) probe; the chained nucleic acid molecule immobilized on a mica substrate is scanned by AFM to take an AFM image of the nucleic acid molecule; the probe is caused to have access to the nucleic acid; and a laser beam is irradiated thereon. Then, a local near field is formed at the probe tip and caused to excite the nucleic acid. The Raman scattered light emitted by the excited nucleic acid is spectroscopically measured to obtain the Raman scattering spectrum of the nucleic acid. Since the S/N ratio of the resulting signal is larger than the number of the bases contained in the nucleic acid, such sensitivity that enables monobasic measurement, is achieved. This method has an advantage in that since the Raman scattering spectrum provides two-dimensional information of a wavenumber vs. intensity pattern, the information volume thereof is exponentially larger than that of one-dimensional information provided by current blockade or tunneling current, exhibiting high ability to identify the bases in qualitative analysis. The size of the near field depends on the curvature of the probe tip and the spatial resolution according to the third example is about 10 nm. Since this value is equivalent to about 30 bases in terms of the number of the bases contained in a nucleic acid, the result obtained in this way contains overlapped information for a multiple of bases. To determine the information for the individual bases from the overlapped information, such a method, for example, has been proposed that a step of scanning along the chain of the nucleic acid with a probe and a step of inferring the bases entering and going out from the near field based on a variance (difference) in spectrum are repeated to obtain sequence information (hereinafter, simply referred to as a “difference method”). To implement this method, it is required that: the nucleic acid is immobilized on the solid state substrate in advance; a high resolution AFM device including high precision stage for measurement is installed; and such a delicate operation that an AFM probe is three-dimensionally scanned at the precision of sub-nm is performed. In other words, the second method has a problem of complicated device construction and operation.